KR20060120715A - Compounds that are active against muscarinic receptors - Google Patents

Abstract

The present invention relates to a compound having activity against a muscarinic receptor,

The compounds and methods may have a beneficial effect by modifying muscarinic m1 receptor activity, alleviating or treating a disease or condition, wherein a therapeutically effective amount of a selective muscarinic m1 functional compound in the method is required for a patient in need of such treatment. It is characterized by being administered.

Description

COMPOUNDS WITH ACTIVITY ON MUSCARINIC RECEPTORS

1 is a graph showing raw data from one 96-well microtiter plate subjected to the screening of 35,000 small organic molecules in the assay described in Example VI

FIG. 2 is a graph showing data comparing profiles of reference antagonist atropine with cells transfected with m1 muscarinic receptor stimulated by carbacol (white triangle) or Compound A (Example I) (black triangle).

The present invention relates to compounds having selectivity for muscarinic acetylcholine receptor subtypes, as well as methods for activating muscarinic receptors and methods for beneficially treating or alleviating diseases with modifications of muscarinic receptor activity.

Muscarinic acetylcholine receptors play a central role in not only the peripheral parasympathetic nervous system but also the central cognitive function. Five other muscarinic receptor subtypes (ie m1-m5) have been established by cloning (TI Bonner et al., Science 237, 1987, pp. 527-532; TI Bonner et al., Neuron 1, 1988, pp. 403-410). m1 is a subtype frequently found in the cerebral cortex and is thought to be involved in controlling cognitive function, m2 is found in the heart and is thought to be involved in controlling heart rate, and m3 is known as sweating and saliva secretion. In addition to being involved in the gastrointestinal tract and urinary stimulation, it has been found that m4 is present in the brain, and m5 is present in the brain and is involved in specific functions of the central nervous system related to the dopaminergic system.

Animal experiments with several muscarinic ligands (S. Iversen, Life Sciences 60 (Nos. 13/14), 1997, pp. 1145-1152) have shown that muscarinic compounds have a significant effect on cognitive functions such as learning and memory. Showed. This suggests that muscarinic agonists may be effective in improving cognitive function in diseases associated with age-related cognitive impairments (such as Alzheimer's disease or other dementia) and age-related cognitive impairments (such as attention deficit hyperactivity disorder). .

Based on the presence of muscarinic receptor subtypes present in other tissues, the m1 receptor subtype is a more abundant subtype in the cerebral cortex, cerebral basal ganglia and hippocampus, and 35-60% of the total of the muscarinic receptor binding site (See A. Levey, Proc. Natl. Acad. Sci. USA 93, 1996, pp.13541-13546). It plays a major role as a post-synaptic muscarinic receptor in the m1 (and possibly m4) subtype (located in acetylcholine-soluble neurons in the neocortex and hippocampus) in several cognitive and motor functions, as measured in the brain region. It seems to play a central role in the m1 response.

Symptoms associated with cognitive impairment such as Alzheimer's disease have already been known to be due to the selective loss of acetylcholine in the brain. This is thought to be the result of degeneration of cholinergic neurons in the basal basal ganglia that dominate the areas of the associated cortex and hippocampus involved in advanced processing (see S. Iversen, supra). The findings suggest that the symptoms can be treated or at least ameliorated by drugs that exhibit cholinergic function in the affected areas of the brain.

Acetylcholine in the brain that indirectly stimulates the muscarinic receptor by treating it with an acetylcholine esterase (AChE) inhibitor, such as 9-amino-1,2,3,4-tetrahydroacridine (tacrine) This increases. Tacrine treatment has been found to improve cognitive function moderately and temporarily in Alzheimer's disease patients (Kasa et al., See above). Tacrine, on the other hand, has been shown to have cholinergic side effects due to peripheral acetylcholine stimulation. This includes abdominal cramps, nausea, vomiting, diarrhea, anorexia, weight loss, myopathy and congestion. Gastrointestinal side effects were found in about one third of patients treated. Tacrine is also known to cause severe hepatotoxicity and elevated liver transaminase has been observed in about 30% of patients (P. Taylor, "Anticholinergic Agents", Chapter 8 in Goodman and Gilman: The Pharmacological Basis of Therapeutics, et al. 9, 1996, pp. 161-176). The side effects of tacrine greatly limit its clinical use. Another AChE inhibitor, (R, S) -1-benzyl-4- [5,6-dimethoxy-1-indanon-2yl] methylpiperidine.HCl (donepezil) has recently been shown to Moderate Alzheimer's disease has been applied as a therapeutic (see P. Kasa et al., Supra). Liver damage was not found in this compound, but gastrointestinal side effects were found similarly to tacrine, which is thought to be due to the stimulation of m3 receptor due to the increased activity of parasympathetic nerves.

Since muscarinic m1 receptors remain unaffected in the frontal cortex and hippocampus, administration of drugs that act as agonists to the muscarinic receptors can cure or at least ameliorate acetylcholine loss in Alzheimer's patients. Already suggested (see JH Brown and P. Taylor, "Muscarinic Receptor Agonists and Antagonists", Chapter 7 in Goodman and Gilman: The Pharmacological Basis of Therapeutics, 9th edition, 1996, p. 147).

The muscarinic agonists (believed to be selective for m1) presented here for the treatment of Alzheimer's disease such as arecholin showed no greater efficacy in clinical trials than AChE inhibitors (see SVP Jones et al., Above). . In one study (T. Sunderland et al., Brain Res. Rev. 13, 1988, pp. 371-389), arecoline significantly improves motor function, elevates mood, and significantly reduces anergia. It has been shown to show no improvement in cognitive function other than the effects on behavioral changes often found in Alzheimer's disease patients. However, it was later found that the m1 agonist and the compound under consideration are selectively weak partial agonists for the m2 and / or m3 receptor subtypes (H. Brauner-Osborne et al., J. Med. Chem. 38, 1995, pp.2188-2195). As described above, vascular actions such as tachycardia and bradycardia found on its agonists are believed to be due to m2 subtype selectivity, and gastrointestinal side effects by their agonists are due to m3 activity.

Therefore, m2 and / or m3 activity is a major problem for muscarinic agonists that have been proposed to treat Alzheimer's disease so far, and greatly limits the dose of drug that can be administered to a patient. In addition, the lack of subtype selectivity and effectiveness of the cholinergic compounds currently being tested has undesirable peripheral side effects, and the effects on cognitive function are limited because of weak and / or adverse effects in the brain. Therefore, it is very beneficial to develop compounds with improved selectivity for the m1 subtype with little or no action on the m2 and m3 subtypes.

The present invention provides a compound having a muscarinic agonist activity of Formula 1, or a pharmaceutically acceptable salt, ester or prodrug thereof:

In Chemical Formula 1,

X ₁ , X ₂ , X ₃ , X ₄ and X ₅ are selected from the group consisting of C, N and O;

k is 0 or 1;

t is 0, 1 or 2;

R ₁ is straight or branched chain, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 alkylidene, C _1-8 alkoxy, C _1-8 heteroalkyl, C _1-8 aminoalkyl, C _1-8 haloalkyl, C _1-8 alkoxycarbonyl, C _1-8 hydroxyalkoxy, C _1-8 hydroxyalkyl, -SH, C _1-8 alkylthio, -O -C (0) -C _5-6 aryl substituted with -CH ₂ -C _5-6 aryl, C _1-3 alkyl or halo; C _5-6 aryl or C _5-6 cycloalkyl, optionally comprising one or more heteroatoms selected from N, S and O; -C (O) NR ₃ R ₄ , -NR ₃ R ₄ , -NR ₃ C (O) NR ₄ R ₅ , -CR ₃ R ₄ , -OC (O) R ₃ ,-(O) (CH ₂ ) _s NR ₃ R ₄ or-(CH ₂ ) _s NR ₃ R ₄ ; Wherein R ₃ , R ₄ and R ₅ are the same or different and each independently comprise one or more heteroatoms selected from the group consisting of H, C _1-6 alkyl, and N, O and S, halo or C C _5-6 aryl optionally substituted with _1-6 alkyl; C _3-6 cycloalkyl group; Or R ₃ and R ₄ form a cyclic cyclic structure comprising 5-6 atoms selected from the group consisting of C, N, S and O together with the N atoms when N atoms are present;

A is C _5-12 aryl or C _5-7 cycloalkyl optionally containing one or more heteroatoms selected from the group consisting of N, S, and O;

R ₂ is H, amino, hydroxyl, halo, or straight or branched C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkoxy, C _1-6 heteroalkyl , C _1-6 aminoalkyl, C _1-6 haloalkyl, C _1-6 alkylthio, C _1-6 alkoxycarbonyl, -CN, -CF ₃ , -OR ₃ , -COR ₃ , NO ₂ , -NHR ₃ , -NHC (O) R ₃ , -C (O) NR ₃ R ₄ , -NR ₃ R ₄ , -NR ₃ C (O) NR ₄ R ₅ , -OC (O) R ₃ . -C (O) R ₃ R ₄ , -O (CH ₂ ) _q NR ₃ , -CNR ₃ R ₄ or-(CH ₂ ) _q NR ₃ R ₄ , where q is an integer from 1 to 6;

n is 0, 1, 2, 3 or 4, and when n> 1, the R ₂ groups are the same or different;

p is 0 or an integer from 1 to 5;

Y is O, S, CHOH, -NHC (O)-, -C (O) NH-, -C (O)-, -OC (O)-, NR ₇ or -CH = N-, and R ₇ is H or C _1-4 alkyl or absent; And

Z is CR ₈ R ₉ , wherein R ₈ and R ₉ are independently selected from the group consisting of H and straight or branched C _1-8 alkyl.

The present invention also provides a pharmaceutical composition comprising an effective amount of a compound of formula (1).

Also provided is a method of treating a symptom of a disease or condition associated with a reduced level of acetylcholine, comprising administering a therapeutically effective amount of a composition comprising a compound of Formula 1.

In a further embodiment, the present invention provides a method of treating a symptom of a disease or condition associated with increased intraocular pressure, such as glaucoma, the method comprising administering a therapeutically effective amount of a composition comprising a compound of Formula 1 Include.

The present invention preferably has a beneficial effect in the treatment of cognitive impairment of Alzheimer's disease or other conditions associated with a decrease in cognitive function with respect to age, in compounds which exhibit a relatively high selectivity for the m1 receptor subtype relative to other muscarinic subtypes. It is to provide a compound that can avoid the side effects of the drug presented for the above purpose. Compounds exhibiting this property are isolated by screening for m1-m5 receptor subtypes.

According to one embodiment, the present invention provides a compound of Formula 1, a pharmaceutically acceptable salt, ester or prodrug thereof:

In Chemical Formula 1,

X ₁ , X ₂ , X ₃ , X ₄ and X ₅ are C; Or one of X ₁ , X ₂ , X ₃ , X ₄ or X ₅ is O or N and the other is C;

k is 0 or 1;

t is 1;

R ₁ is straight or branched chain, C _1-8 alkyl, C _2-8 alkenyl, C _2-8 alkynyl, C _1-8 alkylidene, C _1-8 alkoxy, C _1-8 aminoalkyl, C _1-8 haloalkyl, C _1-8 alkoxycarbonyl, -C (O) NR ₃ R ₄ , -NR ₃ R ₄ , -NR ₃ C (O) NR ₄ R ₅ , -OC (O) R ₃ Or-(CH ₂ ) _s NR ₃ R ₄ , wherein R ₃ , R ₄ and R ₅ are the same or different and are each independently selected from the group consisting of H and C _1-6 alkyl; s is 1 to Is an integer of 8);

n is 1, 2 or 3;

A is phenyl or naphthyl;

R ₂ is straight or branched chain, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkoxy, C _1-6 aminoalkyl, C _1-6 haloalkyl, C _1-6 alkoxycarbonyl, -CN, -CF ₃ , -OH, -COR ₃ , -NHR ₃ , -NHC (O) R ₃ , -C (O) NR ₃ R ₄ , -NR ₃ R ₄ , -NR ₃ C (O) NR ₄ R ₅ , -OC (O) R ₃ or-(CH ₂ ) _q NR ₃ R ₄ , where q is an integer from 1-6; or

A is aryl containing one or more heteroatoms selected from the group consisting of N, S and O;

R ₂ is H, halo, or straight or branched, C _1-6 alkyl, C _2-6 alkenyl, C _2-6 alkynyl, C _1-6 alkoxy, C _1-6 heteroalkyl, C _{1 -6} aminoalkyl, C _1-6 haloalkyl, C _1-6 alkoxycarbonyl, -CN, -CF ₃ , -OH, -COR ₃ , -NHR ₃ , -NHC (O) R ₃ , -C (O ) NR ₃ R ₄ , -NR ₃ R ₄ , -NR ₃ C (O) NR ₄ R ₅ , -OC (O) R ₃ or-(CH ₂ ) _q NR ₃ R ₄

In one preferred embodiment, the compound is a compound of formula

Preferred embodiments of compounds of Formula 2 include compounds of Formulas 2a and 2b:

According to a preferred series of embodiments of the compounds of Formulas 1, 2, 2a and 2b, t is 1 and Y is -C (O)-, -NHC (O)-, S, O or -OC (O)- to be. In others, X ₃ is C. It is preferred that R ₁ is alkyl, where R ₂ is alkyl, aminoalkyl, alkoxy or hydroxyl. In one embodiment p is 3. In others, R ₁ is C _2-8 alkyl and R ₂ is methyl, hydroxyl or alkoxy.

In one embodiment n is 1 or 2; Y is -C (O)-or O and t is 1. R ₂ is preferably halo. According to another embodiment, t is O; Or R ₁ is alkoxy, benzyl or phenyl.

X ₃ is also N, wherein according to one embodiment R ₁ is alkyl or alkoxy; Or R ₁ is benzyl or phenyl; Wherein R ₂ is alkyl or alkoxy.

According to another embodiment, X ₃ is O, where t can be for example O. Preferably, R ₂ is alkyl or alkoxy; Or R ₂ is halo.

Certain embodiments of the present invention include the following components:

4-methoxy-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-ethoxy-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-propoxy-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-butoxy-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-methoxymethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-ethoxymethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-propoxymethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4- (2-methoxyethyl) -1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4- (2-ethoxyethyl) -1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-methoxy-4-methyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-methoxy-4-ethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-methoxy-4-propyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-methoxy-4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-ethoxy-4-methyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-ethoxy-4-ethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-ethoxy-4-propyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-ethoxy-4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-propoxy-4-methyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-propoxy-4-ethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-propoxy-4-propyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-propoxy-4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butoxy-4-methyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butoxy-4-ethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butoxy-4-propyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butoxy-4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

2- [3- (4-n-butylpiperidine) propoxy] toluene;

2- [3- (4-n-butylpiperidine) propanesulfanyl] toluene;

2- [3- (4-n-butylpiperidine) propanesulfinyl] toluene;

3- (4-n-butylpiperidine) -o-tolyl-butan-1-thione;

3- (4-n-butylpiperidinopropyl) -o-tolyl-amine;

N- (4- (4-n-butylpiperidine) -1-o-tolyl-butyl) -hydroxylamine;

4-n-butyl-1- [4- (2-chlorophenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-bromophenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-fluorophenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-mercaptophenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-sulfanylmethylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-sulfanylethylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-aminophenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-methylaminophenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-ethylaminophenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-dimethylaminophenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-diethylaminophenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (1-H-imidazol-2-yl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (1-imidazol-1-yl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (1-thiazol-2-yl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4-([1,2,3] triazol-1-yl) -4-oxo-1-butyl] piperidine;

2- [4-n-butyl-piperidin-1-ethyl] -8-methyl-3,4-dihydro-2H-naphthalen-1-one;

2- [4-n-butyl-piperidin-1-ethyl] -7-methyl-indan-1-one;

3- [4-n-butyl-piperidin-1-ethyl] -chroman-4-one;

2- [4-n-butyl-piperidine-1-ethyl] -1 H-benzoimidazole;

4-n-butyl-1- [4- (4-fluoro-2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-hydroxyphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-methoxyphenyl) -4-oxo-1-butyl] piperidine;

* 4-n-butyl-1- [4- (1-thiophen-2-yl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-ethylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-ethoxyphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2,4-dimethylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2,3-dimethylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (3-methoxyphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-benzyloxyphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (4-methylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-N-phenyl-butyramide;

4-methyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (naphthalen-1-yl) -4-oxo-1-butyl] piperidine;

4-benzyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

1- [4- (2-methylphenyl) -4-oxo-1-butyl] pyrrolidine;

4-benzyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine;

2-propyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

2-ethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-n-propyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine;

3,5-dimethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-methyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine;

4-n-hexyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine;

4-hydroxyethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine;

4-ethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine;

4-benzyl-1- [4- (4-fluorophenyl) -4-oxo-1-butyl] piperidine;

4-benzyl-1- [4- (4-bromophenyl) -4-oxo-1-butyl] piperidine;

4-phenyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine;

3-hydroxymethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-methyl-1- [4- (4-bromophenyl) -4-oxo-1-butyl] piperidine;

1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

2-hydroxymethyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

4-benzyl-1- [4- (2-methylphenyl) -4-oxo-1-pentyl] piperazine;

4-n-hexyl-1- [4- (2-methylphenyl) -4-oxo-1-pentyl] piperazine;

4- (piperidin-1-yl) -1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine;

1- [4- (2-methylphenyl) -4-oxo-1-butyl] -2,3-dihydro-1H-indole;

4-benzyl-1- [5- (2-methylphenyl) -5-oxo-1-pentyl] piperidine;

4-n-butyl-1- [5- (2-methylphenyl) -5-oxo-1-pentyl] piperidine;

4-n-butyl-1- [4- (2,6-dimethylphenyl) -4-oxo-1-butyl] piperidine;

4-n-butyl-1- [4- (2-methoxymethylphenyl) -4-oxo-1-butyl] piperidine;

1- (2-methylphenyl) -2- (4-benzylpiperazin-1-yl) -ethanone;

3,5-dimethyl-1- [5- (2-methylphenyl) -5-oxo-1-pentyl] piperidine;

3,5-dimethyl-1- [4- (4-fluorophenyl) -4-oxo-1-butyl] piperidine;

1- [4- (4-fluorophenyl) -4-oxo-1-butyl] pyrrolidine;

4-benzyl-1- [6- (2-methylphenyl) -6-oxo-1-hexyl] piperazine;

3,5-dimethyl-1- [6- (2-methylphenyl) -6-oxo-1-butyl] piperidine;

4-benzyl-1- [5- (2-methoxyphenyl) -5-oxo-1-pentyl] piperazine;

4-benzyl-1- [3-phenyl-3-oxo-1-propyl] piperazine;

4-n-butyl-1- [5- (2-methoxyphenyl) -5-oxo-1-pentyl] piperidine;

3,5-dimethyl-1- [4- (4-fluoro-2-methylphenyl) -4-oxo-1-butyl] piperidine;

3-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] azetidine;

4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-2-methyl-1-butyl] piperidine;

4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-2,2-dimethyl-1-butyl] piperidine;

4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-2-ethyl-1-butyl] piperidine;

4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-2-propyl-1-butyl] piperidine; And

4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-2,2-diethyl-1-butyl] piperidine.

Compounds specifically excluded in the scope of formula (1) include 4-n-butyl-1- [4-phenyl-4-oxo-1-butyl] piperidine; 4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine; 2- [3- (3-n-butylpiperidine) propanesulfanyl] toluene; And 4-propyloxy-1- [4- (4-fluorophenyl) -4-oxo-1-butyl] piperidine (ie,-(CH ₂ ) _p -Y- is-(CH ₂ ) _3- C (O)-or-(CH ₂ ) ₃ -S- and X ₁ to X ₅ are C; -A- (R ₂ ) _n and R ₁ are absent: o-methyl-phenyl and n, respectively Butyl and phenyl and n-butyl, respectively, or p-fluoro-phenyl and -O- (CH ₂ ) ₂ CH ₃ , respectively.

The invention also provides a method for agonizing a muscarinic receptor comprising reacting an effective amount of a compound of Formula 1 with a receptor, including all compounds within the range of Formula 1 (ie, 4-n-butyl -1- [4-phenyl-4-oxo-1-butyl] piperidine; 4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine; 2- [3- (3-n-butylpiperidine) propanesulfanyl] toluene and 4-propyloxy-1- [4- (4-fluorophenyl) -4-oxo-1-butyl] piperidine ).

The present invention also provides a pharmaceutical composition comprising an effective amount of a compound of Formula 1, including all compounds within the range of Formula 1 (ie, -n-butyl-1- [4-phenyl-4-oxo-1- Butyl] piperidine; 4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine; 2- [3- (3-n-butylpiperidine) propane Sulfanyl] toluene and 4-propyloxy-1- [4- (4-fluorophenyl) -4-oxo-1-butyl] piperidine).

The invention also provides a method of treating a symptom of a disease or condition associated with a reduced level of acetylcholine, a method comprising administering a therapeutically effective amount of a composition disclosed herein. Examples of diseases or disorders are neurodegenerative diseases, cognitive impairments, age related cognitive decline or dementia.

The compounds of the present invention have also been demonstrated in their ability to reduce intraocular pressure and can therefore be used to treat diseases such as glaucoma. Glaucoma is a disease in which abnormalities are found in the circulatory-regulatory mechanism of aqueous fluids that fill the anterior chamber, the space formed between the cornea and the lens. This increases the volume of aqueous fluids, increases intraocular pressure and eventually narrows the field of vision, resulting in vision loss due to forcing and contraction of the papillary of the optic nerve.

Preferably, the compounds of the present invention show selective agonist activity on the m1 receptor. The agonist is defined as a compound that increases the activity of the m1 muscarinic receptor when in contact with the receptor. Selectivity is a property of muscarinic m1 agonists in which the amount of agonist effective for increasing the activity of the m1 receptor hardly increases or increases the activity of the m3 and m5 subtypes and preferably the m2 and m4 subtypes. Is defined.

As used above, the term "alkyl" is straight or branched chain having 1-6 carbon atoms in the chain, such as methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, etc. Mean alkanes. The term "heteroalkyl" is considered to refer to an alkane group containing one or two heteroatoms selected from O, S or N.

As used above, the term "alkenyl" refers to a straight or branched chain alkene group having 2-6 carbon atoms in the chain; The term "alkynyl" is considered to refer to a straight or branched chain alkyne group having 2-6 carbon atoms in the chain.

As used above, the terms "aryl" and "cycloalkyl" are preferably monocyclic and bicyclic ring structures consisting of 5 to 12 carbon atoms, more preferably monocyclic rings consisting of 5 to 6 carbon atoms Means. When the ring consists of one or more heteroatoms selected from N, S and O (ie a heterocyclic ring), the ring consists of 5 to 12 atoms in total, more preferably 5 to 6 atoms. Heterocyclic rings are furyl, pyrroyl, pyrazolyl, thienyl, imidazolyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, pyridyl, piperidinyl, piperazinyl, pyridazinyl, pyri Midinyl, pyrazinyl, morpholinyl, oxadiazolyl, thiadiazoleyl, imidazolinyl, imidazolidinyl, and the like. The ring may be substituted by one or more groups included in the definition of R ₂ above. Substituents C _1-6 alkyl, C _1-6 alkenyl, C _1-6 alkynyl, C _1-6 alkoxy, C _1-6 heteroalkyl, C _1-6 aminoalkyl, C _1-6 haloalkyl or C _{1 -6} alkoxycarbonyl is understood to be substituted by one or more hydroxyl, C _1-4 alkoxy, halogen, cyano, amino or nitro, if present.

As used herein, the term "halogen" or "halo" includes chlorine, fluorine, iodine and bromine.

It is understood that the rings represented by the following structures may both be saturated and unsaturated:

The compounds of the present invention can be prepared by methods analogous to those disclosed in British Patent GB 1,142,143 and US Pat. No. 3,816,433. It will be apparent to those skilled in the art how to modify the method to include other reactive ingredients and the like. Thus, for example, the compound of formula 1 may be prepared as shown in Scheme 1 below.

Starting compounds of formula (VIII) may be prepared by conventional organic synthesis methods. General methods for preparing compounds of formula (VIII) are described in Fuller, RW et al., J. Med. Chem. 14: 322-325 (1971); Foye, WO et al., J. Pharm. Sci. 68: 591-595 (1979); Bossier, JR and many others, Chem. Abstr. 66: 46195h and 67: 21527a (1967); Aldous, FAB, J. Med. Chem. 17; 1100-1111 (1974); Fuller, RW et al., J. Pharm. Pharmacol. 25: 828-829 (1973); Fuller, RW et al., Neuropharmacology 14: 739-746 (1975); Conde, S et al., J. Med. Chem. 21: 978-981 (1978); Lukovits, I. et al., Int. J. Quantum Chem. 20: 429-438 (1981); And Law, B., J. Cromatog. 407: 1-18 (1987), the full text of which is hereby incorporated by reference. Derivatives represented by formula (VII) labeled with radioisotopes can be prepared using reductive amination with trithiated reducing agents or ¹⁴ C-labeled starting materials.

Optionally, the starting compound comprises a carbonyl group, and the compound of formula (VII) is reduced by, for example, AlH ₃ , diborane: methyl sulfide or other standard carbonyl reducing agent to form a ligand of formula (VII).

Receptor ligands of formula (XII) can be prepared by nucleophilic substitution of an electrophile (E) by an amino derivative (XI). Examples of electrophiles that can be used for this purpose include halides such as I, Cl, Br, tosylate or mesylate.

When Y in formula (XII) is -C (O)-, the compound may contain sec-alcohol with pyridinium chlorochromate or N-chlorosuccinimide or CrO ₃ -H ₂ SO ₄ or nickel peroxide or metal (Al , K) or by oxidation with DCC-DMSO.

When Y in formula (XII) is -O-, the compound can be prepared, for example, by alkylating an alcohol with an arylhalide under Cu catalysis.

When Y in formula (XII) is -S-, the compounds can be prepared, for example, by alkylating thiols with arylhalides under Cu catalysis.

When Y in formula (XII) is -CHOH-, the compound can be prepared by catalytic hydrogenation, by the use of NaBH ₄ or by the reduction of the corresponding ketone by the use of LiAlH ₄ .

Suitable pharmaceutically acceptable salts of the compounds of this invention include solutions of pharmaceutically acceptable acids such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid and Acid addition salts that can be prepared by mixing a solution of the compound are included. In addition, when the compound of the present invention includes an acidic moiety, suitable pharmaceutically acceptable salts thereof include alkali metal salts such as sodium salts or potassium salts; Alkaline earth metal salts such as calcium or magnesium salts; And salts formed with suitable organic ligands such as quaternary ammonium salts. Examples of pharmaceutically acceptable salts include acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, carbonate, chloride, clavulanate, citrate, dihydrochloride, fumarate , Gluconate, glutamate, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, mandelate, mesylate, methyl bromide, methylnitrate , Methylsulfate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, phosphate / diphosphate, salicylate, stearate, sulfate, succinate, tannate, tartrate, tosylate, trieti Odide and valerate salts.

The present invention includes within its scope prodrugs of the compounds of the present invention. Typically, the prodrugs are inactive derivatives of the compounds of the present invention that can be readily converted into the required compounds in vivo. Conventional methods for the selection and preparation of suitable prodrug derivatives are described, for example, in "Design of Prodrugs" ed. H. Bundgaard, Elsevier, 1985. Metabolites of such compounds include active substances produced when introducing a compound of the invention into a biological environment.

If the compounds according to the invention have at least one chiral center, they may exist as racemic compounds or as enantiomers. It is to be understood that all such isomers and mixtures thereof are included within the scope of the present invention. And, some of the crystalline forms for the compounds of the present invention may exist as polymorphs, which are also considered to be within the scope of the present invention. In addition, some of the compounds of the present invention may form a solvent compound (ie, a hydrate) having water or a conventional organic solvent. Such solvates are also included within the scope of the present invention.

When a mixture of stereoisomers is obtained by a method for preparing a compound according to the invention, the isomers can be separated by conventional methods such as preparative chiral chromatography. The compounds may be prepared in racemic form, or each enantiomer may be prepared by stereoselective synthesis or resolution. For example, the compounds form salts with optically active acids such as (-)-di-p-toluoyl-d-tartaric acid and / or (+)-di-p-toluoyl-l-tartaric acid. Can be separated into their constituent enantiomers by standard techniques, consisting of the formation of a pair of diastereo isomers followed by fractional crystallization and regeneration of the free base. The compounds may also be separated by the formation of diastereo isomer esters or amides, followed by separation by chromatography and removal of chiral appendages.

In carrying out any of the preparations of the compounds of the invention, it is necessary and / or desired to protect the functional groups or reactors at specific positions in the molecule concerned. This is shown in Protective Groups in Organic Chemistry, ed. J.F.W. McOmie, Plenum Press, 1973; And T.W. Greene & P.G.M. As described in Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991, it can be obtained by conventional protecting groups. The protecting group can be removed in a conventional continuous step using methods known in the art.

The compounds of the present invention may be administered in any of the compositions described above, and may be administered according to the dosing regimen established in the art if a particular pharmacological modification of the activity of the muscarinic receptor is required.

The invention also provides pharmaceutical compositions comprising at least one compound of the invention in combination with a pharmaceutically acceptable diluent or excipient. Preferably, the composition is a tablet, pill, capsule (including sustained-release or delayed-release combination), powder, granule, elixir, tincture, syrup and emulsion, sterile parenteral solution or suspension, aerosol or liquid Sprays, drops, ampoules, auto-injection devices or suppositories for oral, parenteral (eg, intravenous, intramuscular or subcutaneous), intraluminal, sublingual or rectal administration, or by inhalation or aeration And may be formulated according to the examples described in Remington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co., Easton PA, 1990, and by any suitable method. Optionally, the composition is in a sustained-release form suitable for once-weekly or once-monthly administration; For example, insoluble salts of active compounds, such as decanoate salts, are suitable to provide a dipot preparation for intramuscular injection. The present invention also provides a topical formulation suitable for administration to such places as the eye or skin or mucous membranes.

For example, for oral administration in the form of tablets or capsules, the active drug ingredient can be combined with an oral nontoxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. And, if desired or necessary, suitable binders, lubricants, disintegrating agents, flavoring agents and coloring agents may be introduced into the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycols, waxes, and the like. Lubricants used in such dosage forms include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

To prepare a solid composition, such as a tablet, the active ingredient is mixed with a suitable pharmaceutical excipient such as above and other pharmaceutical diluents such as water to give a solid pre-form or pharmaceutical composition thereof containing a homogeneous mixture of the compounds of the present invention. Form an ever acceptable salt. The term "homogeneous" means that the active ingredient is evenly dispersed throughout the composition so that the composition can be easily re-dissolved into the same effect unit dosage form such as tablets, pills and capsules. The solid pre-formulation can then be re-dissolved into unit dosage forms of this form containing from 0.1 to about 50 mg of the active ingredient of the invention. Tablets or fills of the compositions can be combined to provide a dosage form that is coated or otherwise provides the benefit of extended operating time. For example, tablets or pills may consist of an outer layer as a coating surrounding the center and an inner center containing the active compound. The outer coating is an enteric layer that provides for preventing dissolution in the stomach and allows the inner center to pass through while maintaining its original shape up to the duodenum or delayed release. Many materials include a mixture of conventional acids and polymeric acids and many polymeric acids, such as cholac, cetyl alcohol and cellulose acetate, and can be used in enteric layers or coatings.

Liquid formulations to which the composition is incorporated for administration orally or by injection are flavored emulsions such as cottonseed oil, sesame oil, coconut oil with sieves as well as aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and edible oils. Or peanut oil and similar pharmaceutical carriers. Suitable dispersing or suspending agents in aqueous suspensions include synthetic and natural gums such as tragacanth, acacia, alginate, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose or polyvinyl-pyrrolidone. Other dispersants that may be used include glycerin and the like. For parenteral administration, sterile suspensions and solutions are preferred. Usually, isotonic formulations containing suitable preservatives are used when intravenous administration is desired. The composition may also be prescribed as an ophthalmic solution or suspension formulation, such as eye drops, for ocular administration.

As a result, the present invention also relieves a disease or condition in which a modification of muscarinic receptor activity, in particular m1 receptor activity, has a beneficial effect by administering a therapeutically effective amount of a compound of the invention to a patient in need of such treatment. Or to a method of treatment. The disease or condition may be due to inappropriate stimulation or activation of muscarinic receptors. It is expected that by using compounds selective for certain muscarinic receptor subtypes, in particular m1, it is possible to address the side effects of known muscarinic drugs such as tachycardia or bradycardia or action in the stomach.

The term "subject" as used herein refers to an animal, preferably a mammal, most preferably a human, that is the subject of treatment, observation or experiment.

As used herein, the term "therapeutically effective amount" refers to a biological or medical response comprising alleviating the symptoms of a disease to be treated in a tissue, system, animal or human being investigated by a researcher, veterinarian, doctor or other clinician. Means the amount of an effective compound or agent which represents.

Preferably, the compound of formula 1 exhibits subtype selectivity for the muscarinic m1 receptor subtype. Likewise, the compounds exhibit selectivity for muscarinic m1 receptor subtypes compared to other human G-protein coupled receptors including serotonin, histamine, dopamine or adrenergic receptors. One important point of this selectivity is that the compound is effective in treating or improving various diseases and disorders of the central nervous system without the undesirable side effects already found by non-selective compounds.

In the nature of the compounds of the invention capable of demonstrating muscarinic m1 receptor subtype selectivity, the compounds thereof are forms of attention deficit disorder, neurodegenerative diseases such as Alzheimer's disease, and other forms of cognitive decline associated with age such as senile dementia. Or cognitive impairment such as motor impairment, mood swings, anergy, lethargy, instability and symptoms related to dementia with aggressive behavior. Since muscarinic m1 receptors are also involved in regulating intraocular pressure, it is believed that muscarinic m1 agonists can be used to treat or alleviate eye diseases such as glaucoma.

The compound of the present invention may be administered in a single daily administration, or the total daily administration may be divided into two, three or four times a day. In addition, the compounds of the present invention may be administered in the form of thickening by topical use or via the subcutaneous route, using topical thickening excipients using the form of subcutaneous skin patches known to those skilled in the art. To be administered in the form of a subcutaneous delivery system, dosage administration will be continuous rather than intermittent through the dosing schedule.

Dosage regimens using the compounds of the present invention may include a variety of factors including the type, race, age, weight, sex and medical condition of the patient; Severity of the disease to be treated; Route of administration; Kidney and hepatic function of the patient; And the specific compound used. A physician or veterinarian of ordinary skill can readily determine and prescribe an effective amount of the drug necessary to arrest, inhibit or stop the progression of the disease or condition to be treated.

The daily dosage of the product varies over a wide range of 0.01 to 100 mg per day for adults. For oral administration, the composition is provided to the patient to be treated in the form of a tablet containing 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0 or 50.0 mg of the active ingredient for symptom adjustment of the administration. It is desirable to be. Unit administration usually contains from about 0.001 mg to about 50 mg of active ingredient, preferably from about 1 mg to about 10 mg of active ingredient. An effective amount of medicament is usually supplied at a dosage level of about 0.0001 mg / kg to about 25 mg / kg of body weight per day. Preferably, the range is about 0.001 to about 10 mg / kg of body weight per day and in particular about 0.001 mg / kg to 1 mg / kg of body weight per day. The compound is administered on a schedule of 1 to 4 times per day.

The compounds according to the invention can be used alone at appropriate dosages obtained by general tests to obtain optimal pharmacological effects on muscarinic receptors, in particular muscarinic m1 receptor subtypes, with no toxic or other unwanted effects. Suppress to the minimum. And co-administration or sequential administration of other agents that improve the effect of the compound is desirable in some cases.

The pharmaceutical properties and selectivity of the compounds of the present invention for specific muscarinic receptor subtypes can be carried out by various other assays using recombinant receptor subtypes, such as conventional second messenger or binding assays. It is preferable to use a human receptor if available here. Specific conventional functional assay systems include receptor selection and amplification assays described in US Pat. No. 5,707,798, which utilize the property of cells transfecting receptor DNA encoding other muscarinic subtypes to amplify in the presence of ligands of the receptor. Methods for screening for compounds with physiological activity are described. Cell amplification is detected by increased levels of markers that cells also express.

The invention is described in more detail in the following examples, which do not limit the scope of the claimed invention.

Example  One

4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine (5)

1-benzyl-4-n-butylidenepiperidine (2). Fill a 500 ml three-necked flask with stirrer with sodium hydride (1.61 g, 67 mmol) and DMSO (40 ml). The resulting suspension is heated to 90 ° C. for 30 minutes until hydrogen evolution stops. The suspension is cooled in an ice-bath for 20 minutes and a slurry of butyltriphenylphosphonium bromide (26.6 g, 67 mmol) in DMSO (70 mL) is added. The red mixture is stirred at room temperature for 15 minutes. 1-benzyl-4-piperidone (1) (14.0 g, 74 mmol) is added slowly over 30 minutes and the mixture is stirred at room temperature overnight. H ₂ O (200 mL) is added to the reaction mixture and extracted with heptane (4 × 100 mL) and ethyl acetate (2 × 100 mL). The combined organic phases are dried and evaporated to dryness, yielding 38.1 g of yellow oil. The oil was distilled to yield 14.9 g (88%) of (2), bp 101-105 ° C. (0.1 mmHg). ¹ H NMR (CDCl ₃ ) δ 0.90-0.95 (t, 3H), 1.25-1.41 (m, 2H), 1.90-2.20 (m, 2H), 2.18-2.30 (m, 4H), 2.40-2.45 (m , 4H), 2.50 (s, 2H), 5.17 (t, 1H), 7.20-7.42 (m, 5H).

4-n-butylpiperidine (3). To a 500 ml flask equipped with a stirrer, a slurry of 10% palladium and (2) (13.2 g, 58 mmol) on charcoal (1.2 g) in ethanol (70 ml) is added and concentrated hydrochloric acid (1.5 ml) is added. . Air is removed from the reaction flask and hydrogen is added through the reaction flask. A total of 2.5 dm 3 of hydrogen is consumed. The reaction mixture is filtered and evaporated, the residue is dissolved in H ₂ O (40 mL) and NaOH (20 mL, 2M) and extracted with ethyl acetate (3 × 100 mL). The combined organic phases are washed with brine (30 mL) and evaporated to dryness to yield 7.1 g of crude product (3). The crude product was CC treated (eluent: heptane: EtOAc (4: 1)) to give pure (3) (2.7 g, 33%) ¹ H NMR (CDCl ₃ ) δ 0.85 (t, 3H). , 1.0-1.38 (m, 9H), 1.65 (dd, 2H), 2.38 (s, 1H), 2.55 (dt, 2H), 3.04 (dt, 2H).

4- (4-n-butylpiperidin-1-yl) butanenitrile (4). (3) (2.3 g, 16.4 mmol) in acetonitrile (20 ml), 4-bromobutyronitrile (2.4 g, 16.4 mmol), potassium carbonate powder (2.5 g, 18 mmol) in a 100 ml flask with magnetic stirrer Put it. The reaction mixture is stirred at room temperature for 5 hours and H ₂ O (15 mL) is added. The mixture is extracted with ethyl acetate (3 x 30 mL) and the combined organic phases are evaporated to dryness to give 3.9 g of crude product (4). The crude product was CC treated (eluent: heptane: EtOAc (1: 1)) to give pure (4) (2.3 g, 87%) ¹ H NMR (CDCl ₃ ) δ0.82 (t, 3H ), 1.19-1.37 (m, 9H), 1.64-1.75 (d, 2H), 1.84-2.01 (m, 4H), 2.39-2.54 (m, 4H), 2.89-2.97 (d, 2H).

4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine (5). 25 ml oven-dried flasks are filled with Mg turnings (125 mg, 5.2 mmol) which are activated by the use of a heat-gun. Under an inert atmosphere, a suspension of 2-iodoanisol (1.13 g, 5.2 mmol) in Et ₂ O (4 mL) is added and the reaction mixture is left at room temperature for 1 hour. Compound (4) (720 mg, 3.4 mmol) dissolved in Et ₂ O (4 mL) was added and the mixture was refluxed overnight. THF (15 mL) and sulfuric acid (4 mL, 2M) are added, the reaction mixture is stirred for 4 hours and NaOH (6 mL, 2M) is added. The reaction mixture is extracted with ethyl acetate (3 x 50 mL) and the combined organic phases are evaporated to dryness to give 1.2 g of crude product (5). The crude product was CC treated (eluent: CH ₂ Cl ₂ : CH ₃ OH (99: 1)) to give pure (5) (0.42 g, 26%) ¹ H NMR (CDCl ₃ ) δ0. 83 (t, 3H), 1.20-1.42 (m, 9H), 1.65-1.73 (d, 2H), 1.96-2.20 (m, 4H), 2.53 (t, 2H), 3.02-3.17 (m, 4H), 3.89 (s, 3H), 6.95-7.01 (m, 2H), 7.44 (t, 1H), 7.65 (d, 1H).

Example 2

3-hydroxymethyl- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine (7)

4- (3-hydroxymethyl-piperidin-1-yl) -butyronitrile (6). Piperidine-3-yl-methanol (1.12 g, 10 mmol) in acetonitrile (10 mL) was added to an oven-dried 25 mL flask, potassium carbonate (1.38 g, 10 mmol) and 4-bromobutyronitrile. (0.90 mL, 9 mmol) is added. The reaction mixture is stirred at room temperature for 12 hours. The mixture is filtered and evaporated to dryness. H ₂ O (20 mL) was added, extracted with ethyl acetate (3 × 20 mL) and the combined organic phases dried (MgSO ₄ ) and evaporated to dryness, requiring further purification in the synthesis of compound (7). 1.50 g of crude product (6) which can be used without or obtained.

3-hydroxymethyl- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine (7). To an oven-dried 50 mL flask is added Mg turning (780 mg, 32 mmol) activated using a heat-dry under vacuum and dry THF (7 mL). Under an inert atmosphere, a suspension of 2-iodotoluene (5.3 g, 24 mmol) in THF (10 mL) is added and the reaction mixture is refluxed for 4 hours. A suspension of compound 6 (1.50 g, 8 mmol) in THF (5 mL) was added via syringe, CuBr (23 mg, 0.16 mmol, 2 mol%) was added and the reaction mixture was stirred at room temperature overnight. do. The reaction mixture was quenched by addition of H ₂ SO ₄ (20 mL, 2M), stirred at room temperature for 2 hours and then NaOH (8 mL, 2M). THF (15 mL) is added, extracted with CH ₂ Cl ₂ (3 × 20 mL), the organic phase is dried (MgSO ₄ ), evaporated to dryness, and 0.41 g of crude product (7) is produced. The crude product was subjected to preparative HPLC CC treatment [eluent: buffer A: 0.1% TFA; Buffer B: 80% CH ₃ CN + 0.1% TFA], pure sample for analysis of compound (7) is generated. LC-MS [M + H] ⁺ 275 (cald. 275.2).

Example 3

2-propyl- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine (9)

4- (2-propyl-piperidin-1-yl) -butyronitrile (8). 2-propyl piperidine (550 mg, 4.3 mmol), 4-bromobutyronitrile (430 mg, 3.0 mmol) and potassium carbonate (550 mg, 4.0 mmol) in acetonitrile (5 mL) were indoors for 12 hours. Stir at temperature and add saturated brine (25 mL). The reaction mixture is extracted with ethyl acetate (3 × 25 mL) and the combined organic phases are dried (MgSO ₄ ) and evaporated to dryness to yield crude product (8). The crude product was CC treated [eluent: CH ₂ Cl ₂ : MeOH (99: 1)] to yield pure (8) (0.48 g, 83%); LC-MS [M + H] ⁺ 194 (cald. 194.2).

2-propyl- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine (9). To an oven-dried 10 ml flask is added Mg turning (97 mg, 4.1 mmol) which is activated using a heat-dry under vacuum. Under an inert atmosphere, a suspension of 2-iodotoluene (380 μl, 2.8 mmol) in Et ₂ O (3 mL) is added and the reaction mixture is refluxed for 1 hour. A mixture of compound (8) (0.43 g, 2.2 mmol) in CH ₂ Cl ₂ (3 mL) is added via syringe and the reaction mixture is stirred at room temperature overnight. The reaction mixture was quenched by addition of H ₂ SO ₄ (10 mL, 2M), stirred at room temperature for 12 hours and then NaOH (10 mL, 2M). THF (15 mL) was added, extracted with ethyl acetate (3 × 50 mL) and the combined organic phases washed with brine (10 mL) and NaOH (10 mL, 2M), dried (MgSO ₄ ) and dried Evaporation back yields 0.43 g of crude product 9. The crude product was subjected to preparative HPLC [eluent: buffer A: 0.1% TFA; Buffer B: 80% CH ₃ CN + 0.1% TFA], a pure sample for analysis of compound (9) is generated. LC-MS [M + H] ⁺ 287 (cald. 287.2).

Example 4

1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperidine (11)

To an oven-dried 10 ml flask is added Mg turning (97 mg, 4.1 mmol) which is activated using a heat-dry under vacuum. Under an inert atmosphere, a suspension of 2-iodo-toluene (380 μl, 3.0 mmol) in Et ₂ O (3 mL) is added and the reaction mixture is refluxed for 1 hour. Of 4-piperidin-1-yl-butanenitrile (10) (Dahlbom et al. Acta. Chem. Scand. 1951, 5, 690-697) (0.305 mg, 2.0 mmol) in CH ₂ Cl ₂ (3 mL). The suspension is added via syringe and the reaction mixture is stirred at room temperature overnight. The reaction mixture is cooled by adding H ₂ SO ₄ (10 mL, 2M), stirred at room temperature for 12 hours and then NaOH (12 mL, 2M). THF (15 mL) was added, extracted with ethyl acetate (3 × 50 mL) and the combined organic phases washed with brine (10 mL) and NaOH (10 mL, 2M), dried (MgSO ₄ ) and dried Evaporation back yields 0.21 g of crude product 11. The crude product was subjected to preparative HPLC [eluent: buffer A: 0.1% TFA; Buffer B: 80% CH ₃ CN + 0.1% TFA], resulting in a pure sample for analysis of compound 11; LC-MS [M + H] ⁺ 245 (cald. 245.2).

Example 5

4-Methyl-1- [4- (4-bromophenyl) -4-oxo-1-butyl] piperidine (12)

To a 10 ml dried flask was added 4-methylpiperidine (719 μl, 6 mmol), dioxane (5 mL), potassium carbonate (0.30 g, 2.18 mmol), potassium iodide (10 mg) and 4-bro Parent-4-chlorobutyrophenone (785 mg, 2.76 mmol) is added. The reaction mixture is left at 110 ° C. for 12 h and diluted with H ₂ O (10 mL). The reaction mixture is extracted with Et ₂ O (3 × 15 mL) and the combined organic phases are dried (MgSO ₄ ) and evaporated to dryness to give 0.50 g of crude product 12. The crude product was subjected to preparative HPLC [eluent: buffer A: 0.1% TFA; Buffer B: 80% CH ₃ CN + 0.1% TFA], resulting in a pure sample for analysis of Compound (12); LC-MS [M + H] ⁺ 322 (cald. 323.1).

Example 6

1- [4- (2-methylphenyl) -4-oxo-1-butyl] pyrrolidine (13)

An oven-dried 10 ml flask is charged with Mg turning (30 mg, 1.2 mmol) activated using a heat-gun under vacuum. Under an inert atmosphere, a solution of 2-iodotoluene (0.22 g, 1.0 mmol) in Et ₂ O (2 mL) is added and the reaction mixture is refluxed for 1 hour. A mixture of 4-pyrrolidin-1-yl-butyronitrile (J. Org. Chem. 1961, 26, 4070-4076) (0.14 g, 1.0 mmol) in CH ₂ Cl ₂ (2 mL) It is added via syringe and the reaction mixture is stirred at room temperature overnight. The reaction mixture is quenched by adding H ₂ SO ₄ (10 mL, 2M), stirred at room temperature for 2 hours and then NaOH (10 mL, 2M) is added. THF (15 mL) is added, extracted with ethyl acetate (3 × 20 mL) and the organic phase is dried (MgSO ₄ ) and evaporated to dryness to yield 0.12 g of crude product 13. The crude product was subjected to preparative HPLC [eluent: buffer A: 0.1% TFA; Buffer B: 80% CH ₃ CN + 0.1% TFA], pure sample for analysis of compound (13) was generated. LC-MS [M + H] ⁺ 231 (cald. 231.3).

Example 7

4-methyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine (15)

4- (4-Methyl-piperazin-1-yl) -butyronitrile (14). 1-methyl-piperazine (0.52 g, 5.1 mmol), 4-bromobutyronitrile (0.78 g, 5.3 mmol) and potassium carbonate (0.71 g, 5.3) suspended in acetonitrile (5 mL) in a 25 mL flask mmol). The reaction mixture is stirred at room temperature for 4 hours, H ₂ O (20 mL) is added and extracted with ethyl acetate (3 × 25 mL). The combined organic phases are washed with brine (25 mL) and dried (MgSO ₄ ) and evaporated to dryness to yield 0.72 g of crude product 14 which can be used without further purification in the synthesis of compound 15. .

4-Methyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine (15). To a 10 ml oven-dried flask is added Mg turning (116 mg, 4.0 mmol) which is activated using a heat-gun under vacuum. Under an inert atmosphere, a mixture of 2-iodotoluene (0.65 g, 3.0 mmol) in Et ₂ O (3 mL) is added and the reaction mixture is refluxed for 1 hour. A solution of compound 14 (0.33 g, 2.0 mmol) in CH ₂ Cl ₂ (3 mL) is added via syringe and the reaction mixture is stirred at room temperature overnight. The reaction mixture is quenched by the addition of H ₂ SO ₄ (6 mL, 2M) and stirred at room temperature for 2 hours before adding NaOH (8 mL, 2M). THF (15 mL) is added and extracted with CH ₂ Cl ₂ (3 × 20 mL). The organic phase is dried (MgSO ₄ ) and evaporated to dryness to yield 0.26 g of crude product 15. The crude product was subjected to preparative HPLC [eluent: buffer A: 0.1% TFA; Buffer B: 80% CH ₃ CN + 0.1% TFA], a pure sample for analysis of Compound (15) is generated. LC-MS [M + H] ⁺ 260 (cald. 260.4).

Example 8

4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine (17)

* 4- (4-Butyl-piperazin-1-yl) -butyronitrile (16). 1-butyl-piperazine (712 mg, 5.0 mmol), 4-bromobutyronitrile (779 mg, 5.3 mmol) and potassium carbonate (687 mg, 5.0) suspended in acetonitrile (5 mL) in a 25 mL flask. mmol). The reaction mixture is stirred at room temperature for 12 hours, H ₂ O (20 mL) is added and extracted with ethyl acetate (3 × 25 mL). The combined organic phases were washed with brine (25 mL) and dried (MgSO ₄ ) and evaporated to dryness to yield 0.89 g of crude product (16) which could be used without further purification in the synthesis of compound (17). do.

4-n-butyl-1- [4- (2-methylphenyl) -4-oxo-1-butyl] piperazine (17). In a 10 ml oven-dried flask is placed Mg turning (100 mg, 4.0 mmol) activated using a heat-gun under vacuum. Under an inert atmosphere, a suspension of 2-iodotoluene (0.66 g, 3.0 mmol) in Et ₂ O (3 mL) is added and the reaction mixture is refluxed for 1 hour. A suspension of compound 16 (0.43 g, 2.0 mmol) in CH ₂ Cl ₂ (3 mL) is added via syringe and the reaction mixture is stirred at room temperature overnight. The reaction mixture is quenched by the addition of H ₂ SO ₄ (6 mL, 2M) and stirred at room temperature for 2 hours before adding NaOH (8 mL, 2M). THF (15 mL) is added, extracted with CH ₂ Cl ₂ (3 × 20 mL) and the organic phase is dried (MgSO ₄ ) and evaporated to dryness to yield 0.50 g of crude product 17. The crude product was subjected to preparative HPLC [eluent: buffer A: 0.1% TFA; Buffer B: 80% CH ₃ CN + 0.1% TFA], pure sample for analysis of compound (17) is generated. LC-MS [M + H] ⁺ 302 (cald. 302.5).

Example 9

4-n-butyl-1- [4- (2-ethoxyphenyl) -4-oxo-1-butyl] piperidine (18)

To an oven-dried 10 ml flask is added Mg turning (94 mg, 3.8 mmol) which is activated using a heat-dry under vacuum. Under an inert atmosphere, a suspension of 1-ethoxy-2-iodobenzene (0.71 g, 2.9 mmol) in Et ₂ O (3 mL) is added and the reaction mixture is refluxed for 3 hours. Compound 4 (0.40 g, 1.9 mmol) dissolved in CH ₂ Cl ₂ (3 mL) is added and the mixture is stirred at 40 ° C. for an additional 3 hours. The reaction mixture was quenched by addition of H ₂ SO ₄ (10 mL, 2M), stirred at room temperature overnight and then NaOH (20 mL, 2M) was added until basic conditions were achieved. The reaction mixture is extracted with ethyl acetate (3 x 50 mL), the combined organic phases are washed with brine (10 mL) and NaOH (10 mL, 2M), and the combined organic phases are dried (MgSO ₄ ), Evaporation to dryness yields 0.60 g of crude product 18. The crude product was CC treated [eluent: Tol: EtOAc (1: 1)] to give pure compound 18 (0.32 g, 34%); LC-MS [M + H] ⁺ 331 (cald. 331.5).

Example 10

4-n-butyl-1- [4- (2,3-dimethylphenyl) -4-oxo-1-butyl] piperidine (19)

To an oven-dried 10 ml flask is added Mg turning (94 mg, 3.8 mmol) which is activated by using a heat-gun under vacuum. Under an inert atmosphere, a suspension of 1-iodo-2,3-dimethylbenzene (0.69 g, 3.0 mmol) in Et ₂ O (5 mL) is added under natural reflux and the reaction mixture is refluxed for 4 hours. A suspension of compound 4 (0.41 g, 2.0 mmol) in CH ₂ Cl ₂ (2 mL) is added to the reaction mixture and left overnight at room temperature. The reaction mixture was quenched by the addition of H ₂ SO ₄ (7 mL, 2M), stirred at room temperature for 3 hours and then NaOH (20 mL, 2M) was added until the base condition was reached. The reaction mixture is extracted with ethyl acetate (3 x 50 mL), the combined organic phases are washed with brine (10 mL) and NaOH (10 mL, 2M), the organic phase is dried (MgSO ₄ ) and evaporated to dryness 0.69 g of crude product 19 are produced. The crude product was CC treated [eluent: CH ₂ Cl ₂ : MeOH (99: 1)] to give pure compound 19 (0.40 g, 64%); LC-MS [M + H] ⁺ 315 (cald. 315.5).

Example 11

4-n-butyl-1- [4- (2,4-dimethylphenyl) -4-oxo-1-butyl] piperidine (20)

Oven-dried 10 ml flasks are charged with Mg turning (95 mg, 3.9 mmol) activated using a heat-gun under vacuum. Under an inert atmosphere, a suspension of 1-iodo-2,4-dimethylbenzene (0.69 g, 2.9 mmol) in Et ₂ O (4.5 mL) is added under natural reflux and the reaction mixture is refluxed for 3 hours. Compound 4 (0.41 g, 2.0 mmol) dissolved in CH ₂ Cl ₂ (2 mL) is added to the reaction mixture under inert atmosphere and stirred at room temperature overnight. The reaction mixture is quenched by addition of H ₂ SO ₄ (8 mL, 2M), stirred at room temperature for 4 hours, and then the reaction mixture is basified by addition of NaOH (20 mL, 2M). THF (20 mL) was added, extracted with ethyl acetate (3 x 50 mL), the combined organic phases were washed with brine (10 mL) and NaOH (10 mL, 2M) and the organic phase dried (MgSO ₄ ). Evaporation to dryness yields 0.61 g of crude product 20. The crude product was CC treated [eluent: CH ₂ Cl ₂ : MeOH (99: 1)] to give pure compound 20 (0.21 g, 35%); LC-MS [M + H] ⁺ 315 (cald. 315.5).

Example 12

4-n-butyl-1- [4- (2-methoxyphenyl) -4-oxo-1-butyl] piperidine (21)

An oven-dried 10 ml flask is charged with Mg turning (0.12 g, 4.9 mmol) activated using a heat-gun under vacuum. Under an inert atmosphere, a suspension of 1-bromo-2-ethylbenzene (0.66 g, 3.6 mmol) in Et ₂ O (2 mL) is added and the reaction mixture is refluxed for 2 hours. A suspension of compound 4 (0.50 g, 2.4 mmol) in CH ₂ Cl ₂ (2 mL) is added via syringe and the reaction mixture is stirred at room temperature overnight. The reaction mixture was quenched by the addition of H ₂ SO ₄ (14 mL, 2M), stirred at room temperature for 2 hours and then NaOH (20 mL, 2M). THF (20 mL) is added, extracted with ethyl acetate (3 x 50 mL), the combined organic phases are washed with brine (10 mL) and NaOH (10 mL, 2M), the organic phase is dried (MgSO ₄ ) And evaporation to dryness yields 0.75 g of crude product 21. The crude product was CC treated [eluent: CH ₂ Cl ₂ : MeOH (99: 1)] to give pure compound 21 (0.68 g, 90%); LC-MS [M + H] ⁺ 315 (cald. 315.5).

Example 13

4-n-butyl-1- [4- (2,4-dimethylphenyl) -4-oxo-1-butyl] piperidine (22)

Oven-dried 10 ml flasks are charged with Mg turning (88 mg, 3.6 mmol) activated using a heat-gun under vacuum. Under an inert atmosphere, a suspension of 1-iodo-2-methoxymethylbenzene (0.67 g, 2.7 mmol) in Et ₂ O (4 mL) is added and the reaction mixture is refluxed for 1 hour. A suspension of compound 8 (0.38 g, 1.8 mmol) in CH ₂ Cl ₂ (4 mL) is added via syringe and the reaction mixture is stirred at room temperature overnight. The reaction mixture was quenched by addition of H ₂ SO ₄ (10 mL, 2M), stirred at room temperature for 2 hours and then NaOH (10 mL, 2M). THF (15 mL) is added, extracted with ethyl acetate (3 x 50 mL), the combined organic phases are washed with brine (10 mL) and NaOH (10 mL, 2M), the organic phase is dried (MgSO ₄ ) Evaporation to dryness yields 0.51 g of crude product 22. The crude product was CC treated [eluent: CH ₂ Cl ₂ : MeOH (99: 1)] to give pure compound 22 (0.14 g, 23%); LC-MS [M + H] ⁺ 331 (cald. 331.5).

Example 14

4-n-butyl-1- [4- (2-pyridinyl) -4-oxo-1-butyl] piperidine (24)

4- (4-Butyl-piperidin-1-yl) -butyric acid methyl ester (23). 4-bromo-butyric acid methyl ester (2.04 g, 11.2 mmol), compound (3) (1.51 g, 10.8 mmol) and potassium carbonate (1.63 g, suspended in CH ₃ CN (10 mL) in a 25 mL reaction flask 11.8 mmol) is added. The reaction mixture is stirred overnight at room temperature, filtered and evaporated to dryness. H ₂ O (50 mL) is added and extracted with ethyl acetate (3 × 100 mL). The combined organic phases are dried (MgSO ₄ ) and evaporated to dryness, resulting in 2.84 g of crude product 23. The crude product is CC treated (eluent: CH ₂ Cl ₂ : MeOH (99: 1)) to give pure compound 23 (1.93 g, 75%). LC-MS [M + H] ⁺ 241 (cald. 241.2).

4-n-butyl-1- [4- (2-pyridinyl) -4-oxo-1-butyl] piperidine (24). To a 25 mL dried reaction flask was added 2-bromopyridine (200 mg, 1.3 mmol) dissolved in CH ₂ Cl ₂ (3 mL) and the temperature was adjusted to -78 ° C. After stirring for 20 minutes, n-BuLi (0.84 mL, 1.4 mmol) is added under inert atmosphere. After an additional 30 minutes, a solution of compound (23) dissolved in CH ₂ Cl ₂ (2 mL) is added. The reaction mixture is warmed to room temperature for one night before quenching with H ₂ SO ₄ (5 mL, 1M). The reaction mixture is extracted with ethyl acetate (6 × 25 mL) and the combined organic phases are dried (MgSO ₄ ) and evaporated to dryness to yield 0.31 g of crude product 24. The crude product was CC treated [eluent: CH ₂ Cl ₂ : MeOH (10: 1)] to give pure compound 24 (75 mg, 12%); LC-MS [M + H] ⁺ 288 (cald. 288.2).

Example 15

4-n-butyl-1- [4- (2-hydroxyphenyl) -4-oxo-1-butyl] piperidine (27)

1-benzyloxy-2-iodo-benzene (25). 2-iodophenol (1.03 g, 4.7 mmol) and potassium carbonate (0.71 g, 5.2 mmol) are dissolved in dry acetone (10 mL) in a 25 mL oven dried flask. The mixture is stirred for 15 minutes, benzyl bromide (0.61 mL, 5.2 mmol) is added and left overnight at room temperature. H ₂ O (50 mL) was added, extracted with ethyl acetate (3 × 50 mL) and the combined organic phases were dried (MgSO ₄ ) and evaporated to dryness to give 1.7 g of crude product (25). Is generated. The crude product was CC treated (eluent: heptane: EtOAc (9: 1)) to give pure compound 25 (1.2 g, 81%). LC-MS [M + H] ⁺ 310 (cald. 310.0).

4-n-butyl-1- [4- (2-benzyloxyphenyl) -4-oxo-1-butyl] piperidine (26). To 25 ml oven-dried flask is added Mg turning (123 mg, 5.1 mmol) activated using a heat-gun under vacuum. A solution of 1-benzyloxy-2-iodo-benzene 25 (1.18 g, 3.8 mmol) in Et ₂ O (10 mL) is added under inert atmosphere and the reaction mixture is refluxed for 3.5 h. A solution of 4- (4-n-butylpiperidin-1-yl) -butanenitrile (4) (0.53 g, 2.5 mmol) dissolved in CH ₂ Cl ₂ (3 mL) was added and the reaction mixture was 40 Stirred overnight at < RTI ID = 0.0 > The reaction mixture is quenched by addition of H ₂ SO ₄ (10 mL, 2M), stirred for 1 hour, and NaOH (20 mL, 2M) is added until basic conditions are achieved. The reaction mixture is extracted with ethyl acetate (3 × 50 mL), the combined organic phases are washed with brine (10 mL) and NaOH (10 mL, 2M), and the combined organic phases are dried (MgSO ₄ ) and dried Evaporation back yields 1.28 g of crude product 26. The crude product was CC treated [eluent: Tol: EtOAc (1: 1)] to give pure compound 26 (0.51 g, 51%); LC-MS [M + H] ⁺ 393 (cald. 393.7).

4-n-butyl-1- [4- (2-hydroxyphenyl) -4-oxo-1-butyl] piperidine (27). 4-n-butyl-1- [4- (2-benzyloxyphenyl) -4-oxo-1-butyl] blood dissolved in dry EtOH (10 mL) and concentrated HCl (0.1 mL) in a 25 mL reaction flask A solution of ferridine 26 (49 mg, 1.2 mmol) is added and palladium is added on charcoal (40 mg). The reaction flask is then filled with H ₂ using a balloon technique and stirred at room temperature overnight under H ₂ atmosphere. The reaction mixture is basified by addition of NaOH (2 mL, 2.0 M) and filtered through celite. The aqueous phase is extracted with ethyl acetate (3 x 50 mL) and the combined organic phases are washed with brine (10 mL) and NaOH (10 mL, 2M), dried (MgSO ₄ ) and evaporated to dryness to give crude 0.42 g of product 27 is produced. The crude product was CC treated [eluent: CH ₂ Cl ₂ : MeOH (99: 1)] to give pure compound 27 (0.21 g, 58%); LC-MS [M + H] ⁺ 303 (cald. 303.2).

Example 16

Screening methods for test compounds in assays using muscarinic receptor subtypes m1, m2, m3, m4 and m5

Transfection of cells with muscarinic receptor DNAs (general method)

NIH 3T3 cells (available as ATCC CRL 1658 from the American Type Culture Collection) contain 4.5 g / l glucose, 4 mM glutamine, 50 units / ml penicillin, 50 units / ml streptomycin (Advanced Biotechnologies, Gathersburg, Maryland). Cultured at 37 ° C. in a humidified (5% CO ₂ ) incubator in Dulbecco's Improved Eagle medium (DMEM) with the addition of 10% calf serum (Sigma, St. Louis, MI) Let's do it. The cells are treated with trypsin-EDTA, centrifuged and incubated 2 × 10 ⁶ per 15 cm dish in 20 ml of DMEM containing 10% calf serum.

The m1-m5 muscarinic receptor subtypes are cloned in the same manner as described in Science 237, 1987, 527 to Bonner et al., And Neuron 1, 1988, 403 to Bonner et al. For m2 and m4 receptors, the cells are transfected together with DNA encoding a chimera between the Gq protein and the five carboxy-terminal amino acids of the Gi protein (Gq-i5 constructs are described in Conklin et al. Nature 363, 1993, page 274). As described in).

On day 1 the cells are transfected using a Superfect transfection reagent (available from Valencia Qiagen, Calif.) According to the manufacturer's instructions. Receptor DNA, β-gal DNA (pSI-β-galactosidase from Madison Promega, Wisconsin), chimeric Gq-i5 DNA for m2 and m4 receptor subtype analysis, and sperm DNA from salmon as filler (St., Michigan Lewis's Sigma) is added to a total of 20 μg of DNA per plate. Prior to addition to the plate, 60 μl of superfect is added to the DNA and the pipette is shaken up and down several times to mix thoroughly. The mixture is incubated at room temperature for 10-15 minutes. The medium is aspirated and 12 ml of fresh DMEM containing 10% calf serum and 50 units / ml penicillin / streptomycin are added to the plate. The DNA-superfect solution is once more mixed into a pipette and added to the stirred plate so that the DNA mixture is evenly distributed on the surface. The cells are incubated at 37 ° C. and 5% CO ₂ overnight.

After incubation, the medium is aspirated and the plate washed once with 15 ml of Hank's buffered saline. Stir the plate to wash thoroughly. 20 ml of fresh DMEM supplemented with 10% calf serum and 50 units / ml penicillin / streptomycin are added to the plate. The cells are incubated for 24-48 hours until the plates are 100% fused.

Analysis of NIH 3T3 Cells Transfected with Muscarinic Receptor Subtype (General Method)

DMEM containing 2% Cyto-SF3 is heated at 37 ° C. in water under sterile conditions. A sterile working stock solution of the test compound to be analyzed is prepared by diluting the compound in DMEM to a concentration of 8 × at the final concentration for testing. Compounds to be included in the assay (carbacol) as positive controls are diluted to a concentration of 8 × of the final concentration of DMEM. 50 μl of DMEM containing 2% Cyto-SF3 is added to each well of a 96-well microtiter plate under sterile conditions. Then 16 μl of compound solution is added to the top wells of the plate and the solution is diluted by taking 16 μl of compound solution from the top well and pipetting it into the next row of wells. Although the method is repeated in successive rows of each well, 50 μl of media per se is used for baseline control wells (wells containing medium and cells, but no test compound) and plate control wells (wells containing medium, But no test compound and cells). The plate is then placed in an incubator at 37 ° C. to equilibrate the temperature and pH.

When the cell culture is in 100% density, the medium is aspirated and each plate is washed with 15 ml Hank's buffered saline (HBS). The cells are left in the incubator for about 10-15 minutes until the HBS turns slightly yellow. The HBS is aspirated and 1 ml of trypsin is added to each plate and stirred to cover the plate completely. The edge of the plate taps several times to free the cells. After the cells have been released from the surface, 8 ml of DMEM containing 10% calf serum and 50 units / ml penicillin and 50 units / ml streptomycin are added to inhibit trypsin. The plate is washed with medium and the cells are pipetted into tubes. The cells are centrifuged at 1000 rpm for 5-10 minutes in an IEC Centra CL2 centrifuge (Sorvall). The medium is then carefully aspirated so that the cells are not liberated. The cell pellet is suspended in 1600 μl DMEM containing 10% calf serum and 50 units / ml penicillin and 50 units / ml streptomycin, and 20 ml of DMEM supplemented with 2% Cyto-SF3 is added. An aliquot of 50 μl of the cell suspension is added to the wells of the 96-well microtiter plates prepared above (except for the plate control wells). The plates are then incubated at 37 ° C. and 5% CO ₂ for 4 days.

After incubation, the medium is removed by inverting the microtiter plate and gently shaking it, which is then blotted on blotter paper. 100 μl of chromogen material (3.5 mM o-nitrophenyl-β-D-galactopyranoside, 0.5% nonidet NP-40 in phosphate buffered saline) is added to each well and the plate absorbs the appropriate absorbance at 405 nm. Incubate at 30 ° C. until Absorbance of the baseline and plate control wells is subtracted from all values.

result

Using the general method described above, NIH 3T3 cells are transfected together with DNAs encoding the m1, m3 and m5 receptor subtypes. Compound libraries containing approximately 35,000 small organic compounds (one per well) are selected for receptors by the method described above. 1 shows data from one 96-well plate from the screen. In this plate, two compounds are activated at one or more transfected receptors. In the full screen, it was confirmed that four related compounds show activity. To determine the activation of the receptors on the screen, the compound is tested as described above for transfecting each receptor with isolated cell culture. Only compound A activates the m1 receptor subtype and acts as a potent partial agonist. The response was also lower compared to carbacol, the control compound.

In a further experiment, four compounds were found to selectively activate the m1 receptor without showing significant activity at the m2, m3, m4 or m5 muscarinic receptor. Compound A, the compound with the highest activity, is not an antagonist to the carbacol-induced response of the five muscarinic receptor subtypes.

Compound A is further tested for agonist activity against several other receptors in α-adrenergic receptor subtypes 1D, 1B, 1A, 2A, 2B and 2C, histamine H1 and serotonin 5-HT1A and 5-HT2A subtypes. . The compound does not show great activity in the assay. In the antagonist test, Compound A did not inhibit the response of α-adrenergic receptor subtype 2A, 2B or 2C, or serotonin receptor subtype 5-HT1A or 5-HT2A. As illustrated in FIG. 2, the reaction induced by Compound A is blocked by the muscarinic antagonist atropine as in the reaction induced by the muscarinic agonist carbacol.

Example 17

R-SAT Analysis

R-SAT analysis (incorporated herein by reference, see US Pat. No. 5,707,798) is performed by exposing cells transfected with m1, m3 or m5 receptors to seven compounds at a concentration of 1.5 μM. The cellular response is expressed as a percentage of the maximum response of the cells (defined as response to 10 μM carbacol). The results are shown in the table below.

compound Receptor and concentration m1 1.5μM m3 1.5μM m5 1.5 μM A (Example 1) 107 +/- 9 7 +/- 8 3 +/- 8 B (Example 4) 76 +/- 11 7 +/- 9 -6 +/- 10 C (Example 15) 91 +/- 9 4 +/- 9 0 +/- 12 D (Example 10) 72 +/- 9 13 +/- 7 2 +/- 15 E (Example 11) 42 +/- 13 9 +/- 3 -3 +/- 2 F (Example 12) 65 +/- 9 9 +/- 7 5 +/- 11 G ^* 66 +/- 19 16 +/- 12 7 +/- 11

** 4-n-butyl-1- [4-phenyl-4-oxo-1-butyl] piperidine

As described above, the compound is a selective agonist of the m1 receptor.

The invention described above and claimed herein is not limited to the specific embodiments described above, as the embodiments are intended to illustrate some aspects of the invention. Certain equivalent embodiments are intended to be included within the scope of this invention. Indeed, various modifications of the present invention as well as those described and disclosed above are apparent to those skilled in the art from the foregoing specification. Such modifications are also intended to be included within the scope of the appended claims.

Various documents are cited here, the full text of which is incorporated by reference.

The present invention has a beneficial effect in the treatment of cognitive impairment of Alzheimer's disease or other conditions associated with a decline in cognitive function with respect to age, in compounds which exhibit relatively high selectivity for the m1 receptor subtype compared to other muscarinic subtypes. While having the effect of providing a compound that can avoid the side effects of the drugs presented for the purpose.

Claims (1)

Compounds as described and shown in the specification, or pharmaceutically acceptable salts thereof.

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